Abstract Intervertebral disc (IVD) degeneration and associated back pain place a significant burden on the population. As a multiphasic tissue-complex, the interfaces between the subcomponents of the IVD are integral to its normal function. Despite this, there has been little focus in the research community on a comprehensive evaluation of the interfaces of the IVD during degeneration and regeneration. The first objective of this proposal is to quantitatively evaluate the interfaces between the cartilage endplate-annulus fibrosus (EP-AF) and annulus fibrosus-nucleus pulposus (AF-NP) across multiple length scales in healthy and degenerative discs. This will be accomplished via two Specific Aims. Aim 1 will probe the multi-scale structure-mechanics relationships of the interfacial regions (EP-AF, AF-NP) of healthy human and rabbit IVDs. The interfacial regions of human and rabbit IVDs will be evaluated at the microscale via analysis of biochemical content, histology, scanning electron microscopy (SEM), atomic force microscopy (AFM) and second harmonic generation (SHG) imaging. Macroscale properties will be characterized by MRI (T2 mapping, UTE T2, T1r) and biomechanical testing (in compression and tension). In Aim 2, the impact of disc degeneration on the structure and mechanics of EP-AF and AF-NP interfaces at different length scales will be evaluated. For this Aim, interfacial properties of degenerated rabbit and human discs will be assessed at the micro and macroscale using the techniques outlined Aim 1 in order to elucidate how structure and function change with IVD degeneration. Finally, in Aim 3, these same micro- to macro-scale analyses will be applied to regenerating discs. Our group has advanced biologic total disc replacements (endplate-modified disc-like angle-ply structures, eDAPS) for the treatment of advanced stage degeneration. Translation of these eDAPS will require that the interfacial regions of these engineered discs, between the EP-AF and AF-NP match native tissue benchmarks in order to ensure long-term in vivo function. This Aim will assess the interfacial regions of regenerating discs at multiple length scales and determine how these properties mature in engineered tissues both in vitro and with time post-implantation in a rabbit model of total disc replacement. The results from this study will further our understanding of the structure-function relationships of normal and degenerated interfaces in the IVD, and in engineered tissues. Overall, this work has the potential to significantly improve the diagnosis and clinical treatment of back pain associated with disc degeneration.!